Coenzyme B12 (aka, adenosylcobalamin, AdoCbl) biosynthesis is a major biosynthetic pathway (>25 genes) that is unique to prokaryotes. Given the complex chemistry required to assemble this coenzyme, studies of its biosynthesis offer an opportunity to learn complex enzymology and to investigate how this large pathway is integrated with other metabolic processes in the cell. Coenzyme B12 is exclusively synthesized by prokaryotes, and it is essential to the survival of many pathogens. Hence, precise knowledge of the biochemistry underpinning this major pathway, and an understanding of the structural properties of the enzymes involved, is critical to the design of inhibitors that could be used to target disease-causing bacteria or any other prokaryote of societal importance. We will apply a multifaceted approach to continue our studies of AdoCbl biosynthesis. We propose to do three things: i) an in-depth studies of the recently discovered, Fe-S-containing, oxygen-labile EutT corrinoid adenosyltransferase; ii) we will dissect the function of the CbiZ enzyme, whose role in lower ligand remodeling was recently uncovered by our group; iii) we will characterize one function of a recently-discovered 5,6-dimethylbenzimidazole-riboside salvaging system found in important human pathogens. This part of the proposed work includes the initial analysis of identify the genes that encode the enzymes of the anaerobic of the lower ligand base, which in cobalamin is 5,6-dimethylbenzimidazole, a pathway that remains unexplored. We will continue to collaborate with structural biologists led by Ivan Rayment (Department of Biochemistry, UW-Madison), and with transition-metal spectroscopists led by Thomas Brunold (Chemistry Department, UW-Madison). Together, we will gain molecular insights into the assembly of this complex coenzyme and its physiological roles in all cells. PUBLIC HEALTH RELEVANCE: Coenzyme B12 is exclusively synthesized by prokaryotes, and it is essential to the survival of animals, including humans. Precise knowledge of the biochemistry underpinning the biosynthesis of coenzyme B12, and an understanding of the structural properties of the enzymes involved, is critical to the design of inhibitors that could be used to target disease-causing bacteria or any other prokaryote of societal importance. Recent collaborative efforts between the PI and Swiss researchers highlight the impact of knowledge gained from the studies of coenzyme B12 synthesis in our fight against cancer. A multifaceted approach will be applied to learn more about the conversion of the vitamin to its coenzyme form, to elucidate biosynthetic strategies for the lower ligand base in strict anaerobes, and to reveal how prokaryotes remodel the end product of the pathway to suit their metabolic needs.